Azithromycin and Clarithromycin Inhibition of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

The ID₅₀ values for azithromycin and clarithromycin inhibition of translation and of 50S assembly in Staphylococcus aureus cells have been measured. For clarithromycin, 50% inhibition of growth occurred at 0.075 μg/ml, and the effects on translation and 50S formation were equivalent at 0.15 μg/ml. The inhibition of these processes by azithromycin was less effective, with an ID₅₀ of 2.5 μg/ml for growth and 5 μg/ml for inhibition of translation and 50S formation. The additive effects of each of these drugs on translation and 50S formation account quantitatively for their observed influence on cellular growth rates. In macrolide-treated cells, there was also a direct relationship between the loss of ribosomal RNA from the 50S subunit and its accumulation as oligoribonucleotides. These results are compared with the previously described effects of erythromycin on these same processes. Received: 30 June 1997 / Accepted: 12 August 1997     

Changes in Ribonucleic Acid Turnover During Aerobic and Anaerobic Growth in Rhodopseudomonas spheroides

Differences in the rate and extent of degradation of ribonucleic acid (RNA) labeled by a 30-sec pulse in aerobically or anaerobically grown Rhodopseudomonas spheroides have been studied by using rifampin to block RNA synthesis. In anaerobic cultures, unstable RNA is degraded with a half-life of 1.25 to 2.0 min, and about 40% of the pulse-labeled RNA is stable. In aerobic cultures, the half-life of unstable RNA is increased to 2.5 to 4.0 min, and 50% of the RNA is stable. When aerobic cultures are transferred to anaerobic conditions, there is a rapid drop in half-life and in the proportion of stable RNA. When anaerobic cultures are made aerobic, the reverse changes occur after a lag of about 30 min. Addition of puromycin to either aerobic or anaerobic cultures caused the pulse-labeled RNA to be degraded at the same rate and to the same extent as the RNA in an anaerobic control culture. In contrast, addition of chloramphenicol enhanced the difference in RNA half-life and increased the proportion of stable RNA by about 10% in each case. It is concluded that there is a difference in the stability of an RNA component under aerobic and anaerobic conditions.     

The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

Effect of cordycepin (3''-deoxyadenosine) on virus-specific RNA species synthesized in Newcastle disease virus-infected cells.

Cordycepin (3'-deoxyadenosine) has no effect on the size or relative proportions of Newcastle disease virus-specific 18-22S mRNA species nor on the amount or size of the polyadenylic acid associated with them. Cordycepin does, however, cause an inhibition of incorporation of [3H]uridine into 50S virus-specific RNA relative to 18-22S RNA. This inhibition is probably not a direct effect of the drug on the synthesis of 50S viral RNA. Like cycloheximide, another drug which inhibits 50S RNA accumulation in paramyxovirus-infected cells, cordycepin inhibits protein synthesis as measured by amino acid incorporation. It is likely that the inhibition of 50S RNA accumulation is a secondary effect of protein synthesis inhibition. This is supported by the finding that concentrations of cordycepin and cycloheximide, which inhibit protein synthesis to the same extent, have the same effect on the ratio of 50 to 18-22S virus-specific RNA.     

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Binding studies with the allosteric effector phenylalanine.

The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady-state dialysis. Under most of the experimental conditions examined positive co-operativity was observed for the binding of ligand up to 50% saturation and negative co-operativity above 50% saturation. In the presence of 0.4 M NaCl at pH 8.2 the co-operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine-binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities. The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5'-dithio-bis (2-nitrobenzoic acid) did not bind phenylalanine. The mechanism of co-operativity, the binding of the enzyme to Sepharosyl-phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In constrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNA(F)

The translation of encephalomyocarditis virus (EMC) RNA is markedly inhibited in cell-free systems from interferon-treated, vaccinia virus-infected L-cells (10, 11). The polypeptide products synthesized in response to EMC RNA in cell-free systems from these and untreated infected cells have been analyzed by electrophoresis on polyacrylamide gels. Qualitatively, the same EMC-specific polypeptides were synthesized throughout. In experiments using preincubated microsomes from normal Krebs cells to assay cell sap from L-cells which had been exposed to interferon prior to infection, only the amount of the EMC-specific polypeptide products was reduced. This result suggests that there is an inhibition very early in translation in interferon-treated, infected cells. Initiation seems a priori the more attractive site for this inhibition, but an effect shortly after initiation cannot be excluded. With unfractionated cell-free systems from interferon-treated infected L-cells, however, there appeared to be an additional minor inhibitory effect on polypeptide chain elongation, in that the EMC-specific polypeptides synthesized showed not only a reduction in amount but also a bias towards lower molecular weight. The formylated methionyl initiator tRNA (Fmet-tRNA_F) was used as a further probe into the apparent effect on intiation. With this reagent we have confirmed that there is one major initiation site for the translation of EMC RNA in these cell-free systems. In addition, the results have shown that EMC-specific polypeptide chains initiated with Fmet escape the major interferon-mediated inhibition at or shortly after initiation.     

Effect of proteolytic enzymes on masked insulin receptors in rat submaxillary gland microsomes

Trypsin and alpha-chymotrypsin effects on masked insulin receptors were studied. Phospholipase C treatment, incubation in a high ionic strength buffer or solubilization were used as alternative procedures for the unmasking of insulin receptors. These three methods expose receptor structures which are inaccessible to insulin in the current experimental conditions of binding assays without any significant change in binding affinity. Both exposed and masked receptors proved to be equally sensitive to trypsin and alpha-chymotrypsin degradation. At 25 degrees C, about 5 micrograms trypsin/ml for 50 min or 80 micrograms alpha-chymotrypsin/ml for 200 min were necessary in each case to cause a 50% inhibition of the binding of 125I-iodo insulin to microsomes. The results suggest that masked receptors are only nonfunctional to bind insulin but they are not located in compartments inaccessible to molecules present in the medium.     

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

In vitro activity of the echinocandins. How should it be evaluated?

Echinocandins are a novel class of antifungal drugs. They have good activity against Candida spp and Aspergillus spp. Their low selective toxicity allows their administration at high doses with few secondary side effects. We have reviewed the available data on the endpoints for these drugs in their in vitro susceptibility testing on yeasts and moulds. The microdilution broth method is the most commonly used technique and MIC-1 (80% of growth inhibition) seems to be the most reliable endpoint when yeasts are tested. This endpoint also seems to be the most appropriate for the different drugs when they are combined with echinocandins using the checkerboard method for testing yeasts. By contrast, in the case of moulds, the minimum effective concentration (MEC) correlates better with the in vivo activity than the MIC when echinocandins are tested, and when these drugs are combined with other antifungals, MIC-2 (50% of growth inhibition) seems the most appropriate endpoint. Criteria based on drug pharmacodynamics is the most useful to define the echinocandin endpoints that best correlate with their in vivo efficacy.     

Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF-CEM human T-cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dose range of 10(-5)-10(-4) to CCRF-CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S-phase, while higher concentrations caused cell death with a block at the G1/S-phase interface. These toxic effects are compatible with its previously reported inhibition of the non-haem iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron-dependent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucleotide reductase in CCRF-CEM cells with 5 X 10(-5) M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6-thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF-CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided that RNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.